Project Summary Heterotrimeric guanine nucleotide-binding (G) proteins composed of G?, G?, and G? subunits function as molecular switches in signal transduction. Human G protein pathways are targets of over 30% of clinical drugs and their mutation causes genetic disease, developmental abnormalities, and altered infectious disease susceptibility. While humans have 16 G?, 5 G?, and 12 G? genes, the model plant, Arabidopsis thaliana, has many fewer -- only one canonical G? (GPA1), one G? (AGB1), and three G? (AGG1, AGG2 and AGG3) genes. Yet plant G proteins, as in humans, regulate a multiplicity of pathways. Arabidopsis G protein mutants show developmental defects, defects in pathogen defense, and defects in responses to environmental stresses. While Arabidopsis has only one protein (GCR1) with sequence and structural similarity to the 800+ metazoan GPCRs, Arabidopsis has ~600 receptor-like-kinases (RLKs), which form a monophyletic group with Pelle kinases of metazoans. Mass spectrometric analyses in the PI's lab have revealed multiple RLKs present in GPA1 and AGB1 immunoprecipitates, consistent with new genetic evidence of RLK/G protein interaction and confirmed by our kinase assays showing direct GPA1 phosphorylation by RLKs. Phosphoproteomics identifies G protein subunit phosphorylation at conserved sites in plant and metazoan G protein subunits. The overarching hypothesis of the proposed research is that phosphorylation of G protein subunits is an evolutionarily conserved regulatory mechanism that confers specificity to G protein signaling. Three explicit hypotheses are: 1) phosphorylation of G protein subunits controls G protein guanine nucleotide exchange, GTPase activity and heterotrimer association; 2) ligand-activated kinases (RLKs in plants) are GPCRs at the apex of phospho-controlled G protein signaling networks, and; 3) G? and G?? subunit phosphorylation confers specificity to G protein subunit interaction with downstream partners (effectors). These hypotheses will be tested in the Arabidopsis system by, respectively: 1) biochemical assays of the G protein cycle and protein- protein interactions within the heterotrimer; 2) genetic epistasis analysis between G protein subunits and implicated RLKs, phosphoproteomic analyses of G protein subunits following RLK ligand stimulation and in rlk null lines, and in vitro phosphorylation assays; 3) phenotyping of G protein subunit null mutants complemented with phosphomimic and phosphonull G protein subunits, identification of phosphorylation-dependent subunit- effector interactions by coimmunoprecipitation and protein-protein interaction assays, and targeted tests of effector competition for G protein subunits. Arabidopsis is ideal to address these issues in a systematic and comprehensive manner, as it provides a limited number of G protein subunits, ease of manipulation, a plethora of genetic tools, and an array of whole-organism and single-cell G protein phenotypes. Completion of these goals will reveal new mechanisms of G protein modulation, amenable for manipulation and drug targeting to meet the major challenge of pathway-specific control of G protein signaling for improvement of human health.